Journal:

Article Title: Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli

doi: 10.1128/IAI.71.10.5623-5632.2003

Figure Lengend Snippet: Chemokine induction in Hct-8 cells infected with LEE-negative and LEE-positive STEC strains. Hct-8 cells were infected with the indicated STEC strains (Table ​(Table1).1). (A) At 1 or 4 h, total RNA was extracted from cells and IL-8 and MIP-2α mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in [mRNA] relative to levels at 0 h, and data are shown as the means ± SD for triplicate assays. (B) At 4 h, culture supernatants were collected and assayed for IL-8 protein by ELISA as described in Materials and Methods. Data are shown as the means ± standard errors of the means (SEM) from two experiments. The significance of differences between IL-8 secretion by infected versus uninfected cells is indicated as follows: **, P < 0.001; *, P < 0.05.

Article Snippet: The assay was calibrated using recombinant human IL-8 (R&D Systems).

Techniques: Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli

doi: 10.1128/IAI.71.10.5623-5632.2003

Figure Lengend Snippet: Stimulation of Hct-8 cells by strain EDL933 derivatives. Cells were infected with strains 98NK2, EDL933, EDL933 expressing wild-type or defective copies of the ler gene carried on a multicopy plasmid (designated EDL933 Ler and 95SF2 Ler, respectively), and EDL933 with a deletion mutation in eae (Δeae) (all described previously [23]). (A) At 1 or 4 h, total RNA was extracted from cells and IL-8 and MIP-2α mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in [mRNA] relative to levels at 0 h, and data are shown as the means ± SD for triplicate assays. (B) At 4 h, supernatants were collected and assayed for IL-8 by ELISA. Data are shown as the means ± SEM from two experiments. *, significant difference relative to control cells (P < 0.001).

Article Snippet: The assay was calibrated using recombinant human IL-8 (R&D Systems).

Techniques: Infection, Expressing, Plasmid Preparation, Mutagenesis, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli

doi: 10.1128/IAI.71.10.5623-5632.2003

Figure Lengend Snippet: Induction of IL-8 and MIP-2α mRNA and IL-8 protein in Hct-8 cells treated with purified Stx1 and Stx2. Hct-8 cells were treated with purified Stx1 or Stx2 at the indicated concentrations or with STEC strain 98NK2 or EDL933. (A) At 1 or 4 h, total RNA was extracted from cells and IL-8 and MIP-2α mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in [mRNA] relative to levels at 0 h, and data are shown as the means ± SD for triplicate assays. (B) At 4 h, supernatants were collected and assayed for IL-8 by ELISA. Data are shown as the means ± SEM from two experiments. Significant differences relative to untreated control cells are indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The assay was calibrated using recombinant human IL-8 (R&D Systems).

Techniques: Purification, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli

doi: 10.1128/IAI.71.10.5623-5632.2003

Figure Lengend Snippet: Induction of IL-8 and MIP-2α mRNA and IL-8 protein in Hct-8 cells treated with H21 flagellin. Hct-8 cells were stimulated with H21 flagellin at the indicated concentrations or with STEC strains 98NK2 and EDL933. (A) At 1 or 4 h, total RNA was extracted from cells and IL-8 and MIP-2α mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in [mRNA] relative to levels at 0 h, and data are shown as the means ± SD for triplicate assays. (B) At 4 h, supernatants were collected and assayed for IL-8 by ELISA. Data are shown as the means ± SEM from two experiments. Significant differences relative to untreated control cells are indicated as follows: *, P < 0.001.

Article Snippet: The assay was calibrated using recombinant human IL-8 (R&D Systems).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli

doi: 10.1128/IAI.71.10.5623-5632.2003

Figure Lengend Snippet: Induction of IL-8 and MIP-2α mRNA and IL-8 protein in Hct-8 cells infected with strain 98NK2 stx2 and fliC deletion mutants. Hct-8 cells were stimulated with strain 98NK2, 98NK2Δstx2, or 98NK2ΔfliC. (A) At 1 or 4 h, total RNA was extracted from cells and IL-8 and MIP-2α mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in [mRNA] relative to levels at 0 h, and data are shown as the means ± SD for triplicate assays. (B) At 4 h, supernatants were collected and assayed for IL-8 by ELISA. Data are shown as the means ± SEM from two experiments. *, significant difference relative to strain 98NK2-treated cells (P < 0.01).

Article Snippet: The assay was calibrated using recombinant human IL-8 (R&D Systems).

Techniques: Infection, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal:

Article Title: Enhanced CXC Chemokine Responses of Human Colonic Epithelial Cells to Locus of Enterocyte Effacement-Negative Shiga-Toxigenic Escherichia coli

doi: 10.1128/IAI.71.10.5623-5632.2003

Figure Lengend Snippet: Induction of IL-8 and MIP-2α mRNA and IL-8 protein in Hct-8 cells treated with flagellin from strains 95HE4 and 95ZG1. Hct-8 cells were stimulated with strain 95HE4 (H7) or 95ZG1 flagellin at the indicated concentrations or with STEC strain 95HE4, 95ZG1, or 98NK2. (A) At 1 or 4 h, total RNA was extracted from cells and IL-8 and MIP-2α mRNA was quantitated by real-time RT-PCR. Results are expressed as the fold increase in [mRNA] relative to levels at 0 h, and data are shown as the means ± SD for triplicate assays. (B) At 4 h, supernatants were collected and assayed for IL-8 by ELISA. Data are shown as the means ± SEM from two experiments. Significant differences relative to untreated control cells are indicated as follows: *, P < 0.001.

Article Snippet: The assay was calibrated using recombinant human IL-8 (R&D Systems).

Techniques: Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Onco

Article Title: Angiodrastic Chemokines Production by Colonic Cancer Cell Lines

doi: 10.3390/onco2020006

Figure Lengend Snippet: Figure 3. Comparison of CXCL8 secretion over time by the two cell lines after incubation with cancer or normal serum. Mean and standard error (vertical bars) of five separate experiments are presented.

Article Snippet: Commercially available recombinant human CXCL8, CXCL4, CXCL6, and VEGF were obtained (R&D Systems Inc., Mineapolis, MN, USA) and used for generating standard curves.

Techniques: Comparison, Incubation

Journal: Cell Death & Disease

Article Title: Pivotal role of IL-8 derived from the interaction between osteosarcoma and tumor-associated macrophages in osteosarcoma growth and metastasis via the FAK pathway

doi: 10.1038/s41419-024-06487-y

Figure Lengend Snippet: A Cytokine arrays of 143B-Luc cells, human macrophages, and co-culture CM. B IL-8 production during the co-culture of OS cells (143B-Luc, SJSA-1) and macrophages (THP-1 Mφ, HMDMs). C IL-8 expression in macrophages stimulated with OS-CM. D IL-8 expression in OS cells stimulated with macrophage CM. E UMAP plot of OS lung metastases. F UMAP plot of Iba1, CD163, and IL-8 expression in myeloid cell clusters. G Violin plot depicting IL-8 expression in Iba1 +/− or CD163 +/− myeloid cell clusters. CM conditioned medium, HMDMs human monocyte-derived macrophages, OS osteosarcoma, THP-1 Mφ THP-1-derived macrophage. Data are presented as mean ± standard deviation. **p < 0.01. All data were obtained from at least three independent experiments.

Article Snippet: The reagents used were recombinant IL-8 (208-IL, R&D, MN, USA), mouse IgG1 isotype control (MAB002, R&D), anti-IL-8 antibody (MAB208, R&D), dimethyl sulfoxide (DMSO) (D8418, Sigma-Aldrich, Darmstadt, Germany), and PND-1186 (S7653, Selleck, TX, USA).

Techniques: Co-Culture Assay, Expressing, Derivative Assay, Standard Deviation

Journal: Cell Death & Disease

Article Title: Pivotal role of IL-8 derived from the interaction between osteosarcoma and tumor-associated macrophages in osteosarcoma growth and metastasis via the FAK pathway

doi: 10.1038/s41419-024-06487-y

Figure Lengend Snippet: A Cell viability assay for OS cells treated with or without recombinant IL-8 (rIL-8, 10 ng/mL) using Cell Counting Kit-8. B , C Scratch and invasion assays for OS cells treated with or without co-culture CM. The scratch assay was performed for 12 h, and the invasion assay was conducted for 24 h. Scale bars represent 500 μm. D Cell viability assay for OS cells treated with or without co-culture CM and with or without anti-IL-8 antibodies (IL-8 Abs, 1 µg/mL) using Cell Counting Kit-8. E , F Scratch and invasion assays for OS cells treated with or without co-culture CM and with or without IL-8 Abs (100 ng/mL). The scratch assay was performed for 12 h, and the invasion assay was conducted for 24 h. Scale bars represent 500 μm. CM: conditioned medium, OS: osteosarcoma, ns: not significant. Data are presented as mean ± standard deviation. *p < 0.05 and **p < 0.01. All data were obtained from at least three independent experiments.

Article Snippet: The reagents used were recombinant IL-8 (208-IL, R&D, MN, USA), mouse IgG1 isotype control (MAB002, R&D), anti-IL-8 antibody (MAB208, R&D), dimethyl sulfoxide (DMSO) (D8418, Sigma-Aldrich, Darmstadt, Germany), and PND-1186 (S7653, Selleck, TX, USA).

Techniques: Viability Assay, Recombinant, Cell Counting, Co-Culture Assay, Wound Healing Assay, Invasion Assay, Standard Deviation

Journal: Cell Death & Disease

Article Title: Pivotal role of IL-8 derived from the interaction between osteosarcoma and tumor-associated macrophages in osteosarcoma growth and metastasis via the FAK pathway

doi: 10.1038/s41419-024-06487-y

Figure Lengend Snippet: A Western blot assays for FAK phosphorylation in OS cells treated with co-culture CM and quantification of western blot bands. B Western blot assays for the phosphorylation of FAK (p FAK) in OS cells treated with co-culture CM + isotype IgG (IgG) or + anti-IL-8 antibodies (IL-8 Abs, 1 µg/mL) and quantification of western blot bands. The FAK phosphorylation levels were also compared between the same times. C Cell viability assay for OS cells treated with or without co-culture CM and with or without FAK inhibitor (PND-1186, 1 µM) using Cell Counting Kit-8. D , E Scratch and invasion assay for OS cells treated with or without co-culture CM and with or without FAK inhibitor (PND-1186) (1 µM). The scratch assay was performed for 12 h, and the invasion assay was conducted for 24 h. Scale bars represent 500 μm. CM conditioned medium, FAK focal adhesion kinase, ns not significant, OS osteosarcoma. Data are presented as mean ± standard deviation. *p < 0.05 and **p < 0.01. All data were obtained from at least three independent experiments.

Article Snippet: The reagents used were recombinant IL-8 (208-IL, R&D, MN, USA), mouse IgG1 isotype control (MAB002, R&D), anti-IL-8 antibody (MAB208, R&D), dimethyl sulfoxide (DMSO) (D8418, Sigma-Aldrich, Darmstadt, Germany), and PND-1186 (S7653, Selleck, TX, USA).

Techniques: Western Blot, Co-Culture Assay, Viability Assay, Cell Counting, Invasion Assay, Wound Healing Assay, Standard Deviation

Journal: Cell Death & Disease

Article Title: Pivotal role of IL-8 derived from the interaction between osteosarcoma and tumor-associated macrophages in osteosarcoma growth and metastasis via the FAK pathway

doi: 10.1038/s41419-024-06487-y

Figure Lengend Snippet: A Schematic showing the experimental design for subcutaneous transplantation of OS cells. 143B-Luc cells (2 × 10 6 /mouse) were subcutaneously transplanted in 6–7-week-old BALB/c nu/nu mice, and DMEM or co-culture CM was injected into the para-tumor thrice per week. B Tumor volume with or without co-culture CM measured thrice per week after tumor transplantation. C , D Tumor weight and image of the excised tumor with or without co-cultured CM 14 days after tumor transplantation. E , F Immunohistochemistry and quantification of Ki-67 and phospho-FAK in tumor sections with or without co-culture CM 14 days after tumor transplantation. Two fields of view per sample were randomly selected, and quantification was performed in 10 fields. E Ki-67-positive cells in the field were counted and indicated as Ki-67 labeling index. Scale bars represent 50 μm. G Schematic showing the experimental design for subcutaneous OS transplantation using anti-IL-8 antibodies (IL-8 Abs). H Tumor volume after pretreatment with co-culture CM with or without IL-8 Abs (10 µg/mouse) measured thrice per week after tumor transplantation. I and J Weight and image of the excised tumor pretreated with co-culture CM with or without IL-8 Abs 14 days after tumor transplantation. K , L Immunohistochemistry and quantification of Ki-67 and phospho-FAK in tumor sections with co-culture CM with or without IL-8 Abs 14 days after tumor transplantation. Scale bars represent 50 µm. CM conditioned medium, DMEM Dulbecco’s modified Eagle’s medium, FAK focal adhesion kinase, OS osteosarcoma. Data are presented as mean ± standard deviation; *p < 0.05, **p < 0.01. Each group contained five animals.

Article Snippet: The reagents used were recombinant IL-8 (208-IL, R&D, MN, USA), mouse IgG1 isotype control (MAB002, R&D), anti-IL-8 antibody (MAB208, R&D), dimethyl sulfoxide (DMSO) (D8418, Sigma-Aldrich, Darmstadt, Germany), and PND-1186 (S7653, Selleck, TX, USA).

Techniques: Transplantation Assay, Co-Culture Assay, Injection, Cell Culture, Immunohistochemistry, Labeling, Modification, Standard Deviation

Journal: Cell Death & Disease

Article Title: Pivotal role of IL-8 derived from the interaction between osteosarcoma and tumor-associated macrophages in osteosarcoma growth and metastasis via the FAK pathway

doi: 10.1038/s41419-024-06487-y

Figure Lengend Snippet: A Schematic showing the experimental design for OS tail vein injection. 143B-Luc cells were treated with DMEM or co-culture CM for 12 h prior to tail vein injection; 143B-Luc cells (1 × 10 6 /mouse) were injected into the tail vein of 6–7 week-old BALB/c nu/nu mice. B IVIS imaging and quantification of lung metastasis 14 days after the injection of tumors pretreated with or without co-culture CM. C Hematoxylin and eosin staining of lung sections with or without co-culture CM and quantification of lung colonies. Scale bars represent 1 mm and 500 µm. D Immunohistochemistry of Ki-67 positive cells in lung colonies with or without co-culture CM and quantification of the Ki-67 labeling index. Two colonies per sample were randomly selected, and quantification was performed for 10 colonies. Ki-67-positive cells in the colonies were counted and shown as the Ki-67 labeling index. Scale bars represent 100 µm. E Schematic showing the experimental design for OS tail vein injection using anti-IL-8 antibody. F IVIS imaging and quantification of lung metastasis 14 days after the injection of tumors pretreated with co-culture CM with or without IL-8 antibodies. G Hematoxylin and eosin staining of lung sections with co-culture CM with or without IL-8 Abs and quantification of lung colonies. Scale bars represent 1 mm and 500 µm. H Immunohistochemistry of Ki-67-positive cells in lung colonies with co-culture CM with or without IL-8 Abs and quantification of Ki-67 labeling index. Scale bars represent 100 µm. CM conditioned medium, DMEM Dulbecco’s modified Eagle’s medium, IVIS, in vivo imaging system, OS osteosarcoma. Data are presented as mean ± standard deviation; *p < 0.05, **p < 0.01. Each group contained five animals.

Article Snippet: The reagents used were recombinant IL-8 (208-IL, R&D, MN, USA), mouse IgG1 isotype control (MAB002, R&D), anti-IL-8 antibody (MAB208, R&D), dimethyl sulfoxide (DMSO) (D8418, Sigma-Aldrich, Darmstadt, Germany), and PND-1186 (S7653, Selleck, TX, USA).

Techniques: Injection, Co-Culture Assay, Imaging, Staining, Immunohistochemistry, Labeling, Modification, In Vivo Imaging, Standard Deviation